Hildegund C.J. Ertl

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response

We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 × 1012 vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of ∼8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression*.

MHC class I-cestricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses

The use of E1-deleted recombinant adenoviruses in gene therapy has consistently been associated with transient gene expression and inflammation due to immune-based destruction of the infected cells. We have used murine models of adenovirus-mediated gene transfer to liver to investigate these immunologic mechanisms. Adoptive transfer experiments, as well as studies involving genetic knockout mice, confirmed the original hypothesis that cell-mediated immunity induced by E1-deleted adenovirus destroyed trans-gene-expressing hepatocytes and defined MHC class I-restricted CD8+ cytolytic lymphocytes as the primary immune effectors for hepatocyte destruction. Responses mediated by CD4+ cells per se were insufficient to mediate destruction of hepatocytes in vivo, despite the activation of virus-specific T helper cells of Th1 subsets. A better understanding of the response of the host to in vivo gene therapy is important in evaluating its usefulness in humans.

Manipulation of the immune response to a plasmid-encoded viral antigen by coinoculation with plasmids expressing cytokines

Inoculation of plasmid vectors encoding a viral protein into muscle tissue was shown to result in expression of the transantigen and, consequently, an antiviral immune response. Here, we show that coinoculation of a plasmid expressing the glycoprotein of rabies virus with plasmids encoding mouse cytokines modulated the immune response to the viral protein. Coinoculation with a vector expressing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the B and T helper cell activity to rabies virus, while coinoculation with a plasmid expressing interferon-gamma (IFN gamma) resulted in a decrease of the immune response to the viral antigen.

CD8 + T-cell responses to adeno-associated virus capsid in humans

Hepatic adeno-associated virus (AAV)-serotype 2 mediatedgene transfer results in transgene product expression that is sustained in experimental animals but not in human subjects. We hypothesize that this is caused by rejection of transduced hepatocytes by AAV capsid–specific memory CD8+ T cells reactivated by AAV vectors. Here we show that healthy subjects carry AAV capsid–specific CD8+ T cells and that AAV-mediated gene transfer results in their expansion. No such expansion occurs in mice after AAV-mediated gene transfer. In addition, we show that AAV-2 induced human T cells proliferate upon exposure to alternate AAV serotypes, indicating that other serotypes are unlikely to evade capsid-specific immune responses.

Replication-Defective Vector Based on a Chimpanzee Adenovirus

ABSTRACT An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.

New Insights on Adenovirus as Vaccine Vectors

Adenovirus (Ad) vectors were initially developed for treatment of genetic diseases. Their usefulness for permanent gene replacement was limited by their high immunogenicity, which resulted in rapid elimination of transduced cells through induction of T and B cells to antigens of Ad and the transgene product. The very trait that excluded their use for sustained treatment of genetic diseases made them highly attractive as vaccine carriers. Recently though results showed that Ad vectors based on common human serotypes, such as serotype 5, may not be ideal as vaccine carriers. A recently conducted phase 2b trial, termed STEP trial, with an AdHu5-based vaccine expressing antigens of human immunodeficiency virus 1 (HIV-1) not only showed lack of efficacy in spite of the vaccines immunogenicity, but also suggested an increased trend for HIV acquisition in individuals that had circulating AdHu5 neutralizing antibodies prior to vaccination. Alternative serotypes from humans or nonhuman primates (NHPs), to which most humans lack pre-existing immunity, have been vectored and may circumvent the problems encountered with the use of AdHu5 vectors in humans. In summary, although Ad vectors have seen their share of setbacks in recent years, they remain viable tools for prevention or treatment of a multitude of diseases.

Random migration precedes stable target cell interactions of tumor-infiltrating T cells

The tumor microenvironment is composed of an intricate mixture of tumor and host-derived cells that engage in a continuous interplay. T cells are particularly important in this context as they may recognize tumor-associated antigens and induce tumor regression. However, the precise identity of cells targeted by tumor-infiltrating T lymphocytes (TILs) as well as the kinetics and anatomy of TIL-target cell interactions within tumors are incompletely understood. Furthermore, the spatiotemporal conditions of TIL locomotion through the tumor stroma, as a prerequisite for establishing contact with target cells, have not been analyzed. These shortcomings limit the rational design of immunotherapeutic strategies that aim to overcome tumor-immune evasion. We have used two-photon microscopy to determine, in a dynamic manner, the requirements leading to tumor regression by TILs. Key observations were that TILs migrated randomly throughout the tumor microenvironment and that, in the absence of cognate antigen, they were incapable of sustaining active migration. Furthermore, TILs in regressing tumors formed long-lasting (≥30 min), cognate antigen–dependent contacts with tumor cells. Finally, TILs physically interacted with macrophages, suggesting tumor antigen cross-presentation by these cells. Our results demonstrate that recognition of cognate antigen within tumors is a critical determinant of optimal TIL migration and target cell interactions, and argue against TIL guidance by long-range chemokine gradients.

Novel, Chimpanzee Serotype 68-Based Adenoviral Vaccine Carrier for Induction of Antibodies to a Transgene Product

ABSTRACT An E1-deletion-containing adenoviral recombinant based on the chimpanzee serotype 68 (AdC68) was developed to express the rabies virus glycoprotein. Mice immunized with this construct (AdC68rab.gp) developed antibodies to rabies virus and remained resistant to challenge with an otherwise lethal dose of rabies virus. In naïve mice immunized intranasally, the rabies virus-specific antibody responses elicited by AdC68rab.gp were comparable with regard to both titers and isotype profiles to those induced by an adenoviral recombinant based on human serotype 5 (Adhu5) expressing the same transgene product. In contrast, subcutaneous immunization with the AdC68rab.gp vaccine resulted in markedly lower antibody responses to the rabies virus glycoprotein than the corresponding Adhu5 vaccine. Antibodies from AdC68rab.gp-immunized mice were strongly biased towards the immunoglobulin G2a isotype. The antibody response to the rabies virus glycoprotein presented by Adhu5rab.gp was severely compromised in animals preexposed to the homologous adenovirus. In contrast, the rabies virus-specific antibody response to the AdC68rab.gp vaccine was at most marginally affected by preexisting immunity to common human adenovirus serotypes, such as 2, 4, 5, 7, and 12. This novel vaccine carrier thus offers a distinct advantage over adenoviral vaccines based on common human serotypes.

Effect of Preexisting Immunity to Adenovirus Human Serotype 5 Antigens on the Immune Responses of Nonhuman Primates to Vaccine Regimens Based on Human- or Chimpanzee-Derived Adenovirus Vectors

ABSTRACT In this study we compared a prime-boost regimen with two serologically distinct replication-defective adenovirus (Ad) vectors derived from chimpanzee serotypes C68 and C1 expressing Gag, Pol, gp140, and Nef of human immunodeficiency virus type 1 with a regimen in which replication-defective Ad vectors of the human serotype 5 (AdHu5) were given twice. Experiments were conducted in rhesus macaques that had or had not been preexposed to antigens of AdHu5. There was no significant difference in T-cell responses tested from peripheral blood of the different groups, although responses were overall highest in nonpreexposed animals immunized with the chimpanzee Ad vectors. Preexisting immunity to AdHu5 completely inhibited induction of transgene product-specific antibodies by the AdHu5 vectors without affecting antibody responses to the chimpanzee vectors. Upon euthanasia, T-cell responses were tested from a number of tissues. Preexisting immunity to AdHu5, commonly found in humans, changed the homing pattern of vaccine-induced T cells. In AdHu5-preexposed animals vaccinated with the chimpanzee Ad vectors, frequencies of transgene-specific T cells were higher in spleens than in blood, and in most preexposed animals vaccinated either with AdHu5 vectors or chimpanzee adenovirus vectors, frequencies of such T cells were exceptionally high in livers. The latter results indicate that analysis of T-cell responses solely from blood mononuclear cells of vaccine recipients may not suffice to compare the potencies of different vaccine regimens.

Chimpanzee adenovirus antibodies in humans, sub-Saharan Africa.

Human sera from the United States, Thailand, and sub-Saharan Africa and chimpanzee sera were tested for neutralizing antibodies to 3 chimpanzee adenoviruses. Antibodies were more common in humans residing in sub-Saharan Africa than in humans living in the United States or Thailand. This finding suggests cross-species transmission of chimpanzee adenoviruses.